Statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, potentiate the anti-angiogenic effects of bevacizumab by suppressing angiopoietin2, BiP, and Hsp90α in human colorectal cancer

Because of their need for oxygen and nutrients for survival, mammalian cells are located within 100 to 200 μm of blood vessels—the diffusion limit for oxygen (Folkman, 1971). For multicellular organisms to grow beyond this size, they must recruit new blood vessels by vasculogenesis and angiogenesis. This process is regulated by a balance between pro- and anti-angiogenic molecules and is usually derailed in cancer (Folkman, 2007). Without blood vessels, tumours cannot grow beyond a critical size or metastasise to another organ. Angiogenesis is a complex process that includes endothelial cell proliferation and movement, as well as endothelial cell-mediated degradation of the extracellular matrix (ECM). The multistep process of angiogenesis is essential for cancer progression and metastasis. Vascular endothelial growth factor (VEGF), a critical mediator of angiogenesis, is usually upregulated in cancer (Ferrara, 2002). Bevacizumab, which is a humanised monoclonal immunoglobulin G1 antibody that targets circulating VEGF, has been shown to improve response rates and survival when combined with any first-line or second-line standard cytotoxic chemotherapy regimen in patients with metastatic colorectal cancer (Hurwitz et al, 2004; Giantonio et al, 2007; Grothey et al, 2008; Van Cutsem et al, 2009). However, bevacizumab does not result in marked survival improvements, and further, its clinical activity depends on the presence of specific resistance profiles. To overcome these limitations and also uncover novel therapeutic approaches, it is necessary to elucidate resistance mechanisms and identify agents that potentiate this targeting agent. Statins are widely used as lipid-lowering agents to reduce cardiovascular risk with a favourable safety profile. Statins, such as lovastatin, simvastatin, pravastatin, and atorvastatin, are potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is the rate-controlling enzyme of the mevalonate pathway. Mevalonate is the initial molecule for a diverse array of end products, such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate, both of which are essential substrates for post-translational modifications of Ras and Ras homologue (Rho). Ras and Rho have important roles in the intracellular signal transduction that is responsible for cell growth, proliferation, migration, and survival (Goldstein and Brown, 1990; Casey, 1995). On the basis of the effect of statins on post-transcriptional modifications of Ras and Rho, the antitumour effect of statins has been suggested in various cancer cell lines (Kim et al, 2001; Park et al, 2001; Lee et al, 2006). The relationship between statins and angiogenesis has been investigated, and interestingly, the effects of statins were found to depend on their blood concentration (Weis et al, 2002). At low nanomolar concentrations, statins have pro-angiogenic effects and are regarded as beneficial for treating cardiovascular diseases (Dimmeler et al, 2001; Llevadot et al, 2001; Assmus et al, 2003; Spyridopoulos et al, 2004). On the other hand, at high micromolar concentrations, they have anti-angiogenic activity and may be of special significance for cancer therapy. The anticancer effects of statins have been shown to result from the inhibition of endothelial cell proliferation and migration, as well as the induction of apoptosis (Negre-Aminou et al, 1997; Pirillo et al, 1997; Sato et al, 1998; Kaneta et al, 2003; Muck et al, 2004; Schaefer et al, 2004); however, a limitation in using statins for cancer therapy is that the required doses are too high to be applied to patients. We previously studied the effects of statins at cardiovascular therapeutic doses in cancer cell lines in vitro (Lee et al, 2006, 2011). Our results showed that 0.3 μM lovastatin induced cell senescence in prostate cancer cells (Lee et al, 2006), and that addition of 0.2 μM simvastatin to cetuximab reduced the proliferation of CRC cells carrying KRAS mutations (Lee et al, 2011). In this present study, we investigated whether cardiovascular therapeutic doses of statins affect angiogenesis and potentiate anti-angiogenic effects of bevacizumab in CRC. On the basis of several previous results (Sato et al, 1998; Kaneta et al, 2003; Muck et al, 2004; Schaefer et al, 2004; Lee et al, 2006), we postulated that the statin dose used had little direct effect on endothelial cells; therefore, we focused on the mediators of angiogenesis. The mechanism underlying the anti-angiogenic effects of statins was evaluated by angiogenesis antibody array and proteomics analysis. The effects of drugs on tumour growth were also tested in mouse xenograft models.